Vaccines have been very effective in protecting people from a wide variety of diseases, whether caused by virus, bacteria, or fungus. The ability of vaccines to induce specific protection against such a wide range of pathogenic organisms results from their ability to stimulate specific humoral antibody responses, as well as cell-mediated responses. This invention relates to a process for preparing such vaccines, and particularly to a process for making protein-polysaccharide conjugates that are used in preparing vaccines, immunogens, and other valuable immunological reagents. The invention further relates to the vaccines and other compositions produced from the conjugates made according to the invention.
Certain agents can stimulate an immune response with minimal chemical modifications, for example, tetanus toxoid, which is immunogenic even in the absence of an adjuvant. Other important agents are either non-immunogenic or poorly immunogenic, but they can be converted into immunogenic molecules or constructs, in which form they can induce vigorous immune responses. For example, most polysaccharides are poorly immunogenic in young animals. After they are coupled to proteins, however, the resulting construct becomes immunogenic. For example, immunization with protein-polysaccharide conjugates enables otherwise unresponsive young children to mount an immune response to the polysaccharide component. The conjugation of proteins to polysaccharides converts the polysaccharide from a weakly immunogenic T-cell independent antigen to a T-cell dependent antigen that recruits T-cell help, and thus stimulates heightened immune responses. Note the discussion by J. M. Cruse, et al. (Editors), Conjugate Vaccines, Karger, Basel, (1989); and R. W. Ellis, et al. (Editors), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994). These books are entirely incorporated herein by reference.
Conjugation of a protein and a polysaccharide can provide other advantageous results. For example, it has been found that protein-polysaccharide conjugates enhance the antibody response not only to the polysaccharide component, but also to the protein component. This effect is described, for example, in the dual conjugate patent applications of Mond and Lees, U.S. patent appln. Ser. No. 08/402,565 (filed Mar. 13, 1995) (now abandoned), now U.S. Pat. No. 5,585,100 (issued Dec. 17, 1996); Appln. Ser. No. 08/444,727 (filed May 19, 1995); and Appln. Ser. No. 08/468,060 (filed Jun. 6, 1995) (now abandoned). These patent applications each are entirely incorporated herein by reference. This effect also is described in A. Lees, et al., "Enhanced Immunogenicity of Protein-Dextran Conjugates: I. Rapid Stimulation of Enhanced Antibody Responses to Poorly Immunogenic Molecules," Vaccine, Vol. 12, No. 13, (1994), pp. 1160-1166. This article is entirely incorporated herein by reference.
Noting at least some of the advantageous results obtained using protein-polysaccharide conjugates, researchers have developed various techniques to facilitate coupling of proteins and polysaccharides. Note W. E. Dick, et al., "Glyconjugates of Bacterial Carbohydrate Antigens: A Survey and Consideration of Design and Preparation Factors," Conjugate Vaccines (Eds. Cruse, et al.), Karger, Basel, 1989, beginning at page 48. This article also is entirely incorporated herein by reference. As one example of a protein-polysaccharide coupling technique, the use of organic cyanylating reagents, such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate, also called "CDAP" in this patent application, has been developed. These reagents activate polysaccharides and facilitate coupling of polysaccharides to proteins for conjugate vaccines. The activated polysaccharides can be directly or indirectly coupled to proteins. The use of CDAP and other organic cyanylating reagents is described in the following U.S. Patent and Patent Applications of Andrew Lees: U.S. patent appln. Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned); U.S. Pat. No. 5,651,971; U.S. Pat. No. 5,693,326; and U.S. patent appln. Ser. No. 08/482,666 (filed Jun. 7, 1995) (now U.S. Pat. No. 5,849,301). These U.S. patents and patent applications each are entirely incorporated herein by reference. The use of CDAP also is described in Lees, et al., "Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate For Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents," Vaccine, Vol. 14, No. 3 (1996), pp. 190-198. This article also is entirely incorporated herein by reference.
Other techniques for coupling proteins and polysaccharides also are known. For example, the use of homobifunctional or heterobifunctional vinylsulfones for protein-polysaccharide conjugation is described in U.S. patent appln. Ser. No. 08/852,733 filed on May 7, 1997, (pending) in the name of Andrew Lees. Protein-polysaccharide coupling using uronium salts and haloacyl reagents is described in U.S. Provisional Patent Appin. Nos. 60/041,781 (filed Mar. 24, 1997) and 60/042,379 (filed Apr. 24, 1997), respectively. These patent applications also are entirely incorporated herein by reference.
In the production of protein-polysaccharide conjugates and vaccines, a major cost and time consuming step lies in the separation of the free protein (i.e., the unreacted or non-conjugated protein that is not covalently bound to a polysaccharide) from the conjugated protein-polysaccharide product. This separation, which is also called "fractionation," usually is accomplished using a column chromatographic technique (e.g., size exclusion chromatography or gel filtration) or an ultrafiltration process. These protein separation processes significantly increase the time and expense involved in producing protein-polysaccharide conjugates and vaccines. Under the good manufacturing procedure ("GMP") guidelines, a dedicated (and expensive) chromatography column normally is needed for each type of conjugate to prevent contamination of the product.
In addition to the increased production cost and time, this free protein separation step often results in a significant loss of the desired protein-polysaccharide conjugate material because the conjugate does not easily release from the chromatographic matrix. This factor further increases the costs involved in preparing a protein-polysaccharide conjugate vaccine.
Given the state of the art as described above, there is a need for a simple, quick, and efficient procedure for separating free protein from a mixture that contains free protein and a protein-polysaccharide conjugate.